Molecular characterization of local breeds is a key factor in biodiversity conservation and should be combined with appropriate breeding strategies to improve the production characteristics of indigenous breeds and prevent breed decline. In the current study, we analyzed nine sheep breeds (Czech Wallachian sheep /CVA/, Sumava sheep /SUM/, Improved Wallachian /IVA/, Slovak Wallachian /SVA/, Świniarka /SWI/, Uhruska sheep /UHR/, sheep Podgorska /POG/, sheep Ukraine (UKR), and Tsurcana /TUR/ keep in Carpathia region /Czech Republic, Slovakia, Poland, Ukraine, Romania/ using a set of 13 microsatellite loci. Results showed two main gene pool: CVA and SWI were assigned to a separate gene pool, and the second pool consisted of IVA, SUM, SVA, POG, UHR, UK, and TUR, with a high level of overlap, which suggests that these breeds share a common ancestor and are not clearly differentiated. The set of microsatellite loci may be used further as an effective molecular tool for analyzing genetic diversity.